PROJECT SUMMARY / ABSTRACT Aberrations in lymphatic structure and function are increasingly recognized as a significant source of morbidity in a variety of disease states. For example, lymphatic abnormalities associated with congenital heart disease (CHD) that result in increased pulmonary blood flow (PBF) include congenital or acquired chylothoraces, and immunologic aberrations. Importantly, it is increasingly appreciated that lymphatic abnormalities are not only associated with a variety of pulmonary diseases but participate in their pathogenesis. Recent recognition that abnormal respiratory dysfunction often persists years after cardiac correction and is an independent risk factor for mortality in adult CHD patients has brought renewed urgency to better understand the underlying lymphatic pathobiology in CHD, which thus far remains largely unknown. We have previously used a clinically relevant large animal model of CHD with increased PBF (shunt) in combination with isolated vessel reactivity of the thoracic duct and primary lymphatic endothelial cell (LEC) culture, to demonstrate that chronically increased PBF and the consequential increase in lymphatic flow, is associated with: 1) abnormal pulmonary lymphatic flow and architecture; 2) increased mitochondrial reactive oxygen species (mtROS)-driven hypoxia inducible factor-1 (HIF-1) activity and metabolic reprograming to support cellular and proliferation; and 3) a KLF2-dependent decrease in nitric oxide (NO) signaling. In this renewal application we will test our novel hypothesis that in the setting of increased PBF, the mechanosensory channel Piezo1 plays a pivotal role sensing alterations in lymphatic flow, triggering downstream increases in endothelin-1 (ET-1) and mtROS driven HIF-1a activity. This results in decreased NO bioavailability and subsequent lymphatic dysfunction, that contributes to persistently abnormal respiratory mechanics even after the cardiac defect has been repaired. In support of this hypothesis, we present preliminary evidence demonstrating: 1) increased lymphatic endothelial Piezo1, ET-1, RhoA, and mtROS in shunt LECs; 2) Piezo1-, ET-1-, and mtROS-dependent increases in HIF-1 in control LECs; 3) shunt lambs have abnormal respiratory mechanics and lymphatic endothelial dysfunction that persists following closure of the shunt, despite normalized hemodynamics, and 4) that treatment with a mitochondrially-targeted antioxidant (mitoquinone, MitoQ) reverses the HIF-1 mediated shunt LEC phenotype in vitro and normalizes pulmonary lymphatic architecture and function in vivo. This overall hypothesis will be tested in three inter-related, but independent mechanistic aims, that utilize integrated physiologic, cellular, and molecular experiments. These translational and targetable studies include: 1) whole animal hemodynamic physiologic studies, advanced CT/MR imaging, and sophisticated pulmonary function testing, 2) ex vivo thoracic duct reactivity studies, and 3) transcripti...